1. Field of the Invention
The present invention relates to a novel protein which induces the interferon-xcex3 (hereinafter abbreviated as xe2x80x9cIFN-xcex3xe2x80x9d) production by immunocompetent cells.
2. Description of the Prior Art
It is known that IFN-xcex3 is a protein which has antiviral-, antioncotic- and immunoregulatory-activities and is produced by immunocompetent cells that are stimulated with antigens or mitogens. Because of these biological activities, IFN-xcex3 has been expected for use as an antitumor agent since it was discovered, and studied energetically on clinical trials as a therapeutic agent for malignant tumors in general including brain tumors. IFN-xcex3 preparations commercially available now are roughly classified into two groups, i.e. one group of natural IFN-xcex3s produced by immunocompetent cells and another group of recombinant IFN-xcex3s produced by transformants obtained by introducing DNAs which encode natural IFN-xcex3s into microorganisms of the species Escherichia coli. In the above clinical trials, one of these two groups of IFN-xcex3s is administered to patients as an xe2x80x9cexogenous IFN-xcex3xe2x80x9d.
Among these IFN-xcex3s, natural IFN-xcex3s are usually produced by culturing established immunocompetent cell lines in nutrient culture media admixed with IFN-xcex3 inducers to produce IFN-xcex3s, and purifying the produced IFN-xcex3s from the resulting cultures. It is known that IFN-xcex3 inducers greatly influences on the IFN-xcex3 yield, the facility of IFN-xcex3 purification, and the safety of final IFN-xcex3 preparations. Generally, mitogens such as concanavalin A (Con A), lentil lectin, pokeweed lectin, endotoxin and lipopolysaccharides can be used as IFN-xcex3 inducers. However, these mitogens have the following problems: (i) their molecules and qualities vary and change depending on their origins and purification methods, and (ii) preparations with a constant IFN-xcex3 inducibility are not readily prepared in a satisfactory yield. In addition, most of these mitogens might induce unfavorable side effects when administered to living bodies, and some of them might cause toxicity, so that it is substantially difficult to induce IFN-xcex3 production by directly administering IFN-xcex3 inducers to the living bodies.
The present invention was made based on a novel protein which induces the interferon-xcex3 production by immunocompetent cells. During the study of cytokines produced by mammalian cells, the present inventors noticed that the existence of a substance which induces IFN-xcex3 production in mouse liver cells which had been treated with a lipopolysaccharide and inactivated whole cells of Corynebacterium. They isolated the substance by many purification methods using column chromatography as a main technique and studied the properties and features, and have found that the reality is a protein having the following physicochemical properties:
(1) Molecular weight
xe2x80x8319,000xc2x15,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);
(2) Isoelectric point (pI)
xe2x80x83pI of 4.8xc2x11.0 on chromatofocusing;
(3) Partial amino acid sequence
xe2x80x83Having the partial amino acid sequences of SEQ ID NOS:8 and 9; and
(4) Biological activity
xe2x80x83Inducing the IFN-xcex3 production by immunocompetent cells.
The data concluded that the substance is novel because no protein with these physicochemical properties is known. The present inventors continued studying on mouse liver cells and have succeeded to isolate a DNA which encodes the protein. The inventors decoded the DNA and have found that it consists of 471 base pairs and encodes the amino acid sequence of SEQ ID NO:10 (where the symbol xe2x80x9cXaaxe2x80x9d means xe2x80x9cmethioninexe2x80x9d or xe2x80x9cthreoninexe2x80x9d).
Based on these findings, the present inventors further studied on human liver cells to obtain a DNA which encodes another novel substance that induces the IFN-xcex3 production by immunocompetent cells. They revealed that the reality is a polypeptide, then decoded the DNA and found that it has the amino acid sequence of SEQ ID NO:6 (where the symbol xe2x80x9cXaaxe2x80x9d is xe2x80x9cisoleucinexe2x80x9d or xe2x80x9cthreoninexe2x80x9d). They introduced the DNA into Escherichia coli to express the polypeptide and to produce it in the resulting culture in a satisfactorily high yield. These findings were disclosed in Japanese Patent Laid-Open Nos. 27,189/96 and 193,098/96, applied by the present applicant. In Japanese Patent Application No. 78,357/95 applied by the applicant, the polypeptide is disclosed as an agent for susceptive diseases. Although biologically active proteins which are administered to humans after mixed with pharmaceuticals should be generally human cell origin, no human cell which produces such a polypeptide is reported.
In view of the foregoing, the object of the present invention is to provide a protein of human cell origin, which induces the IFN-xcex3 production by immunocompetent cells.
The another object of the present invention is to provide a process for producing the protein.
The further object of the present invention is to provide the use of the protein as an agent for susceptive diseases.
The first object of the present invention is attained by a protein of human cell origin which induces the IFN-xcex3 production by immunocompetent cells and has the amino acid sequence of SEQ ID NO:1.
The second object of the present invention is attained by a process for producing the protein by propagating human cells which produce the protein, and collecting the protein from the propagated cells.
The third object of the present invention is attained by an agent for susceptive diseases, which contains the protein as an effective ingredient.